Cytometers, such as described in U.S. Pat. No. 5,072,382, issued to applicant herein, have become a common tool for the examination of biological cell samples for various properties and/or defects indicative of abnormalities and diseases. The cytometers utilize a dye absorption property of DNA and of specific DNA sequences (contained in the cells of the samples), with developed procedures, in order to provide a variety of information relating to the DNA content of the cells. These include degree of anomaly, such as variations in DNA content and its character. The information, so obtained, is important in various diagnoses and treatment such as in cancer detection and treatment and in the prior determination of birth defects such as Down's syndrome and the like.
In one developed procedure (using a cytometer), called fluorescent in situ hybridization (FISH), fluorescent dyes (such as fluorescein isothiocyanate (FITC) which fluoresces green when excited by an Argon ion laser) are used to tag a sequence of DNA which is complementary to a defined nucleotide sequence of DNA in the cell, with the sequences being joined, such that cells with specific DNA sequences can be detected microscopically. Each chromosome containing the target DNA sequence will produce a fluorescent spot in every cell, with scanning of the cells with a laser exciting the dye to fluoresce. Thus, for example, specimens hybridized with a DNA sequence known to be contained on chromosome number 21 will produce two fluorescent spots in cells from normal patients and three spots from Down's Syndrome patients because they have an extra chromosome number 21. A microscope based, stationary sample cytometer, used in this procedure, is disclosed in said US Patent issued to applicant, the disclosure of which is incorporated herein by reference thereto.
Typically, thousands of cells are scanned in a cytometric sample and the specific DNA sequence contents are determined in the form of fluorescent spots, which are counted relative to the number of cells. Deviation of the number of spots in a cell from a norm (e.g., such as probing for X chromosomes on human lymphocytes, based on gender--males having one X chromosome and thus normally one fluorescent spot per cell and females having two X chromosomes and thus normally two fluorescent spots per cell) is indicative of a disease, cancer or other abnormality. The relative number of abnormal cells to the total cell sample population is also indicative of the extent of the condition or abnormality.
An initial technique, described in applicants' co-pending application no. 07/987,679, filed Dec. 2, 1992 (the disclosure of which is incorporated herein by reference thereto) for such probe spot counting determination was the differentiation of fluorescing spots within a single cell as opposed to fluorescing spots of adjacent cells. This technique uses measurement of interspot distances, as an accurate means for discrimination, whereby spots within a single cell are always closer than those of even adjacent cells. Thus, the distance between a probe spot and its Nth nearest neighboring spot are measured. If the spots are close, they are on the same cell. If the distance to the Nth nearest neighboring spot is large, they are on different cells. Large populations of cells can be measured thereby and the numbers of cells for each Nth nearest distance value can be plotted. These distributions can thus be used to characterize the number of spots per cell without actually counting spots within the cell boundary.
In a more recently developed method, a second dye, e.g., propidium iodide (PI) has been utilized to contour the nucleus based on PI fluorescence (which fluoresces red when excited by the Argon ion laser) to define the cell boundary, thereby independently contouring the probe spots and counting probe spots within the PI contour as spots defined within the cell.
However, a problem remains, with both methods, as an artifact of the cytometric analysis equipment being utilized. Cytometers, being depth independent, essentially monitor cell samples in a two dimensional plane whereas cells in a sample are in a three dimensional dispersion. As a result, the three dimensional cell is projected on to a two dimensional area. Cells and probe spots therefore often overlay and can not be separated from each other. It is however, important to be able to eliminate the effect of such overlay since in clinical applications, such as with respect to cancer, a deleted gene is diagnostically important. Two factors are present in the overlay problem which must be accounted for. A first factor is the overlay of the cells and the second being an overly of probe spots within a single cell.
It is accordingly an object of the present invention to provide a method for accurately determining the fluorescing spot count of individual cells in a three dimensional sample matrix utilizing a two dimensional monitoring cytometer.
It is a further object of the present invention to be able to separate overlying probe spots for improved diagnostic determinations.
These and other objects, features and advantages of the present invention will become more evident from the following discussion and drawings in which: